This invention is directed to a synthetic biomaterial compound based on stabilized calcium phosphates and more particularly to the molecular, structural and physical characterization of this compound. This compound which in the alternative may be referred to as Skelite(trademark) has applications in the treatment of various bone related clinical conditions such as for the repair and restoration of natural bone compromised by disease, trauma or genetic influences.
Bone is a complex mineralizing system composed of an inorganic or mineral phase, an organic matrix phase, and water. The inorganic mineral phase is composed mainly of crystalline calcium phosphate salts while the organic matrix phase consists mostly of collagen and other noncollagenous proteins. Calcification of bone depends on the close association between the organic and inorganic phases to produce a mineralized tissue.
The process of bone growth is regulated to meet both structural and functional requirements. The cells involved in the processes of bone formation, maintenance, and resorption are osteoblasts, osteocytes, and osteoclasts. Osteoblasts synthesize the organic matrix, osteoid, of bone which after calcium phosphate crystal growth and collagen assembly becomes mineralized. Osteocytes regulate the flux of calcium and phosphate between the bone mineral and the extracellular fluid. Osteoclasts function to resorb bone and are essential in the process of bone remodeling. Disturbing the natural balance of bone formation and resorption leads to various bone disorders. Increased osteoclast activity has been demonstrated to lead to bone disease characterized by a decrease in bone density such as that seen in osteoporosis, osteitis fibrosa and in Paget""s disease. All of these diseases are a result of increased bone resorption.
In order to understand the mechanisms involved which regulate bone cell function, it is important to be able to assess the normal function of bone cells and also the degree of perturbation of this activity in various bone diseases. This will lead to the identification of drugs targeted to restore abnormal bone cell activity back to within normal levels. Together with the identification of the etiology of abnormal and normal bone cell activity and the assessment of this activity, is the desire and need to develop compositions and methods for the treatment of abnormal bone cell activity, as a result of disease, surgical removal or physiological trauma all of which lead to bone tissue loss. Therapeutics which provide for the replacement and repair of bone tissue, such as with the use of bone implants, are highly desired.
Several groups have attempted to provide compositions suitable for the therapeutic replacement of bone tissue. U.S. Pat. No. 4,871,578 discloses a process for the formation of a non-porous smooth coating of hydroxyapatite suitable for implant use. U.S. Pat. No. 4,983,182 discloses a ceramic implant which comprises a sintered body of zirconia and a coating of xcex1-TCP and zirconia, or hydroxyapatite and zirconia. U.S. Pat. No. 4,988,362 discloses a composition for the fusion of a bioceramic to another bioceramic. U.S. Pat. No. 4,990,163 discloses a coating used for the production of bioceramics which consist of xcex1-TCP and xcex2-TCP. Although these different compositions may be used as biocompatible coatings for implants and the like, none of these compositions have been demonstrated to participate in the natural bone remodeling process. Furthermore, none of the prior compositions developed, can be manipulated to reliably produce a range of films, thicker coatings and bulk ceramic pieces which share a common composition and morphology which leads to similar bioactive performance in vivo and in vitro.
It has therefore long been the goal of biomaterials research in the field of orthopedics to develop synthetic structures exhibiting comprehensive bioactivity. Bioactive synthetic substrates and scaffolds capable of incorporation into the natural process of bone remodeling are of interest in applications which include not only in vitro bone cell assays [1], but also in vivo resorbable bone cements [2, 3], implantable coatings which enhance the bonding of natural bone to the implant [4], various forms of implantable prostheses and bone repair agents [5, 6], and ex vivo tissue engineering [7]. The prime objective for such materials in vivo is to combine the stimulation of osteogenic activity in associated bone tissues for optimum healing, with the capability to be progressively resorbed by osteoclasts during normal continuous remodeling [8]. In vitro, related functions are to provide standardized laboratory test substrates on which osteoclast resorptive function or osteoblast production of mineralized bone matrix can be assessed and quantified [1]. Such substrates must be stable and insoluble in the biological environment until acted upon by osteoclasts, the specific bone mineral resorbing cells.
While calcium hydroxyapatite (Ca5(OH)(PO4)3 or HA) is the primary inorganic component of natural bone [9], trace elements are also present [10]. Calcium hydroxyapatite is but one of a number of calcium-phosphorous (Caxe2x80x94P) compounds which are biocompatible. Others include octacalcium phosphate [11] and both phases of tricalcium phosphate (Ca3(PO4)2 or xcex1-TCP/xcex2-TCP) [12]. Compounds, particularly HA, may show differing degrees of stoichiometry with the Ca/P ratio ranging from 1.55 to 2.2 [13]. Such materials can be artificially created by conventional high temperature ceramic processing [14] or by low temperature aqueous chemistry [15, 16]. Most of such artificial materials show good biocompatibility in that bone cells tolerate their presence with few deleterious effects, and indeed enhanced bone deposition may occur [17, 18]. Currently, the most recognized medical application of calcium phosphates is the coating of implantable prosthetic devices and components by thermal or plasma spray to render the surface osteoconductive. It has been noted that Caxe2x80x94P ceramics which are stable in biological environments are often a mixture of individual compounds [19]. However, despite the osteogenic potential of these artificial materials, none actively participate in the full process of natural bone remodeling.
In an effort to understand the cellular mechanisms involved in the remodeling process, several research groups have attempted to develop methods to directly observe the activity of isolated osteoclasts in vitro. Osteoclasts, isolated from bone marrow cell populations, have been cultured on thin slices of natural materials such as sperm whale dentine (Boyde et al Brit. Dent. J. 156, 216, 1984) or bone (Chambers et al J. Cell Sci. 66, 383, 1984). The latter group have been able to show that this resorptive activity is not possessed by other cells of the mononuclear phagocyte series (Chambers and Horton, Calcif Tissue Int. 36, 556, 1984). More recent attempts to use other cell culture techniques to study osteoclast lineage have still had to rely on the use of cortical bone slices (Amano et al. and Kerby et al J. Bone and Min. Res. 7(3)) for which the quantitation of resorptive activity relies upon either two dimensional analysis of resorption pit areas of variable depth or stereo mapping of the resorption volume. Such techniques provide at best an accuracy of approximately 50% when assessing resorption of relatively thick substrata. In addition these analysis techniques are also very time consuming and require highly specialized equipment and training. Furthermore, the preparation and subsequent examination of bone or dentine slices is neither an easy nor practical method for the assessment of osteoclast activity.
The use of artificial calcium phosphate preparations as substrata for osteoclast cultures has also met with little success. Jones et al (Anat. Embryol 170, 247, 1984) reported that osteoclasts resorb synthetic apatites in vitro but failed to provide experimental evidence to support this observation. Shimizu et al (Bone and Mineral 6, 261, 1989) have reported that isolated osteoclasts resorb only devitalized bone surfaces and not synthetic calcium hydroxyapatite. These results would indicate that functional osteoclasts are difficult to culture in vitro.
In the applicant""s published international PCT application WO94/26972, cell-mediated resorption was shown to occur on a calcium phosphate-based thin film formed by the high temperature processing of a calcium phosphate colloidal suspension on quartz substrates. When used in vitro, these ceramic films exhibited multiple discrete resorption events (lacunae) across their surface as a result of osteoclast activity, with no evidence of dissolution arising from the culture medium. The regular margins of these lacunae correspond closely to the size and shape of the ruffled borders normally produced by osteoclasts as the means by which they maintain the localized low pH required to naturally resorb bone mineral in vivo. Enhanced deposition of mineralized bone matrix also occurs on these ceramics in the presence of osteoblasts.
It is now demonstrated by the Applicant""s that these thin film ceramics exhibit two general characteristics: (1) the presence of a mixture of Caxe2x80x94P containing phases comprising approximately 33% HA and approximately 67% of a silicon stabilized calcium phosphate and (2) a unique morphology. Importantly, it was noted that the thermal processing of the Caxe2x80x94P colloid at 1000xc2x0 C. resulted in an HA powder, while the same colloidal suspension processed on quartz had a mixed HA and silicon stabilized calcium phosphate phase composition. Energy dispersive X-ray analysis of the film demonstrated the presence of Si in the coating while cross-sectional transmission electron microscopy indicated a microporous physical structure.
Applicants have discovered that the presence of stabilizing entities can stabilize the composition and prevent its degradation in physiological fluids. Hence, disappearance of calcium phosphate entities from a film, coating or bulk ceramic piece of this composition, is substantially due to the activity of the osteoclasts and not due to a dissolution process. The stabilized artificial bioactive composition is the first such composition which supports both osteoclast and osteoblast activity and which allows for the reliable assessment of the physiological activities of both cell types as well as for the development of both diagnostic and therapeutic strategies.
In view of the clinical importance of developing a synthetic bone graft that is both osteogenic and can participate in the body""s natural cell-based remodeling process, it was important to focus on the role of introduced additives such as silicon in the formation of a calcium phosphate-based biomaterial compound capable of being assimilated and remodeled into natural bone with the aid of the activity of osteoclasts and osteoblasts. Since the compound could only be characterized by the preparation method, it was crucial to be able to both physically and chemically characterize the compound. In particular, it was important to characterize the physical structure of the compound and more importantly, the specific molecular and chemical structure of the stabilized compound in order to be able to understand why the new compound worked so well in biological conditions affecting the skeleton. The physical, molecular and chemical characterization of the compound could also provide for the development of further uses of the compound in the treatment of several different types of bone-related clinical conditions. In addition, this would also allow further chemical alteration of the compound in order that it could be designed for use in specific in vivo, in vitro and ex vivo applications.
The Applicant""s work now pointed to the transformation of HA into a stabilized calcium phosphate phase. Surprisingly, during the difficult course of explicit characterization of the compound from a molecular standpoint, it was found that the resultant stabilized compound was an entirely new compound herein described and termed Skelite(trademark).
The present invention provides a stabilized composition comprising a synthetic biomaterial compound which allows for a wide variety of diagnostic and therapeutic applications. The biomaterial compound, in accordance with an aspect of the invention, can be used to provide a range of fine or coarse powders, pellets, three-dimensional shaped pieces, thin films and coatings which share a common globular morphology and an interconnected microporosity. In addition, the biomaterial compound can be formed as a macroporous structure in order to provide an artificial three dimensional geometry similar to that found in bone in vivo. The biomaterial compound, made in any form, encourages the activity of bone cells cultured thereon and also allows for the development of ex vivo engineered artificial bone tissues for use as bone grafts.
The created stabilized calcium phosphate compound has only now been specifically characterized with respect to its physical and chemical structure leading to the realization that the stabilized compound was an entirely new compound never before described. The biomaterial compound is made by the high temperature processing of a fine precipitate, formed from a colloidal suspension and stabilized using an additive with an appropriate sized ionic radius that enables substitution into the Caxe2x80x94P lattice. The compound typically coexists with calcium hydroxyapatite and is itself a novel stabilized calcium phosphate compound having a microporous morphology based on inter-connected particles of about 0.2-1.0 xcexcm in diameter. The compound is essentially insoluble in biological media but is resorbable when acted upon by osteoclasts. It also promotes organic bone matrix deposition by osteoblasts and can be assimilated into natural bone during the natural course of bone remodeling through the activity of osteoclasts and osteoblasts. The compound has been extensively analyzed using X-ray diffraction, infrared spectroscopy, nuclear magnetic resonance spectroscopy, and light scattering particle analysis. Results now indicate that the characteristic features of the compound arise during sintering through substitution reactions where a stabilizing element such as silicon enters the calcium phosphate lattice under conditions of high chemical reactivity. The crystallographic features are linked through the glaserite form of the apatite structure.
According to an aspect of the present invention a biomaterial compound is provided comprising calcium, oxygen and phosphorous, wherein at least one of the elements is substituted with an element having an ionic radius of approximately 0.1 to 1.1 xc3x85.
According to another aspect of the present invention is a biomaterial compound having the formula (Ca1-wAw)i[(P1-x-y-zBxCyDz)Oj]2; wherein A is selected from those elements having an ionic radius of approximately 0.4 to 1.1 xc3x85; B, C and D are selected from those elements having an ionic radius of approximately 0.1 to 0.4 xc3x85; w is greater than or equal to zero but less than 1; x is greater than or equal to zero but less than 1; y is greater than or equal to zero but less than 1; z is greater than or equal to zero but less than 1; x+y+z is greater than zero but less than 1; i is greater than or equal to 2 but less than or equal to 4; and j equals 4-xcex4, where xcex4 is greater than or equal to zero but less than or equal to 1.
Specific compounds of the present invention include but are not limited to Ca3(P0.750Si0.25O3.875)2 and Ca3(P0.9375Si0.0625O3.96875)2.
The knowledge of the specific molecular and chemical properties of the compound of the present invention allows for the development of several uses of the compound in various bone-related clinical conditions. Such applications may include orthopedic, maxillo-facial and dental applications where the compound can be fabricated to exist as a fine or coarse powder, pellets, three-dimensional shaped pieces, macroporous structures, thin films and coatings.
According to yet another aspect of the present invention is a method for substituting natural bone at sites of skeletal surgery in human and animal hosts with a biomaterial compound comprising calcium, oxygen and phosphorous wherein at least one of the elements is substituted with an element having an ionic radius of approximately 0.1 to 1.1 xc3x85. The method comprises the steps of implanting the biomaterial compound at the site of skeletal surgery wherein such implantation promotes the formation of new bone tissue at the interfaces between the biomaterial compound and the host, the progressive removal of the biomaterial compound primarily through osteoclast activity, and the replacement of that portion of the biomaterial compound removed by further formation of new bone tissue by osteoblast activity, such progressive removal and replacement being inherent in the natural bone remodeling process.
In accordance with another aspect of the present invention is a method for repairing large segmental skeletal gaps and non-union fractures arising from trauma or surgery in human and animal hosts using a biomaterial compound comprising calcium, oxygen and phosphorous wherein at least one of the elements is substituted with an element having an ionic radius of approximately 0.1 to 1.1 xc3x85. The method comprises the steps of implanting the biomaterial compound at the site of the segmental skeletal gap or non-union fracture wherein such implantation promotes the formation of new bone tissue at the interfaces between the biomaterial compound and the host, the progressive removal of the biomaterial compound primarily through osteoclast activity, and the replacement of that portion of the biomaterial compound removed by further formation of new bone tissue by osteoblast activity, such progressive removal and replacement being inherent in the natural bone remodeling process.
According to yet another aspect of the present invention is a method for aiding the attachment of implantable prostheses to skeletal sites and for maintaining the long term stability of the prostheses in human and animal hosts using a biomaterial compound comprising calcium, oxygen and phosphorous wherein at least one of the elements is substituted with an element having an ionic radius of approximately 0.1 to 1.1 xc3x85. The method comprises the steps of coating selected regions of an implantable prosthesis with the biomaterial compound, implanting the coated prosthesis into a skeletal site wherein such implantation promotes the formation of new bone tissue at the interfaces between the biomaterial compound and the host, the generation of a secure interfacial bond between the host bone and the coating, the subsequent progressive removal of the coating primarily through osteoclast activity such that the coating is diminished, and the replacement of that portion of the biomaterial compound removed by further formation of new bone tissue to generate a secure interfacial bond directly between the host bone and the prosthesis.
According to yet another aspect of the present invention is a method for providing tissue-engineering scaffolds for bone replacement in human or animal hosts using a biomaterial compound comprising calcium, oxygen and phosphorous wherein at least one of the elements is substituted with an element having an ionic radius of approximately 0.1 to 1.1 xc3x85. The method comprises the steps of forming the biomaterial compound as a macroporous structure comprising an open cell construction with interconnected voids, combining mature and/or precursor bone cells with the macroporous structure, and allowing the cells to infiltrate the structure in order to develop new mineralized matrix throughout the structure.
The knowledge of the structure of the novel compound of the present invention also allows for the use of the compound as a carrier for various pharmaceutical agents including but not restricted to bone growth factors and other agents affecting bone growth and remodeling.
According to another aspect of the present invention is a method for delivering pharmaceutical agents to the site of skeletal surgery in human or animal hosts using a biomaterial compound comprising calcium, oxygen and phosphorous wherein at least one of said elements is substituted with an element having an ionic radius of approximately 0.1 to 1.1 xc3x85. The method comprises combining a pharmaceutical agent with the biomaterial compound and applying the pharmaceutical agent combined with the biomaterial compound to a site of skeletal surgery, wherein such application results in controlled local release of the pharmaceutical agent.
The biomaterial compound may be combined with additives such as those which increase the mechanical strength and toughness of the compound in order to provide additional functions for specific applications. The biomaterial compound may also be combined with various calcium phosphate materials such as calcium hydroxyapatite, xcex1-TCP, xcex2-TCP, octocalcium phosphate, tetracalcium phosphate, dicalcium phosphate and calcium oxide either as a physical mixture or as a solid solution.
The biomaterial compound has a distinguishable microporous and nanoporous structure along with a crystallography that is similar yet different from that of xcex1-TCP. The new compound exhibits monoclinic pseudo-rhombic symmetry and is in the monoclinic space group P21/a. Furthermore, the new compound has a portion of the phosphorous substituted by an element having a suitable ionic radius.
The knowledge of the chemical formula of the biomaterial compound and the mechanism behind its bioactivity and stability in biological environments allows for the use of this compound in vivo for the treatment of various bone related clinical conditions. In particular, the compound may be used to help repair and restore natural bone that has been compromised by disease, trauma, or genetic influences.
In accordance with yet a further aspect of the invention is a bioactive synthetic sintered composition for providing a morphology capable of consistently supporting bone cell activity thereon, the composition comprising stabilized calcium phosphate compound developed by the conversion of a hydroxyapatite substance in the presence of stabilizing entities at sintering temperatures wherein the stabilizing entities stabilize and insolubilize the calcium phosphate compound.
In accordance with a further aspect of the present invention is a process for preparing a synthetic sintered composition comprising a stabilized calcium phosphate compound having a morphology suitable for supporting bone cell activity thereon, the process comprising converting a hydroxyapatite substance in the presence of stabilizing entities at sintering temperatures wherein the stabilizing entities stabilize and insolubilize the calcium phosphate compound.
According to yet a further aspect of the present invention is a synthetic sintered microporous polycrystalline structure for supporting bone cell activity, the structure comprising a stabilized calcium phosphate compound having a globular morphology of interconnected rounded particles with an interconnected microporosity in said structure.
In accordance with yet a further aspect of the present invention is an implant comprising: a) a scaffold for supporting the implant; and b) a layer of a stabilized calcium phosphate compound developed by the conversion of a hydroxyapatite substance in the presence of stabilizing entities at sintering temperatures wherein the stabilizing entities insolubilize and stabilize the calcium phosphate compound.
In accordance with another aspect of the present invention is an implant comprising: a) a scaffold for supporting the implant; b) a layer of a stabilized calcium phosphate compound developed by the conversion of a hydroxyapatite substance in the presence of stabilizing entities at sintering temperatures wherein the stabilizing entities insolubilize and stabilize the calcium phosphate compound; c) a boundary layer deposited by osteoblasts cultured on the layer of the stabilized calcium phosphate compound; and d) a mineralized collagenous matrix secreted by such cultured osteoblasts.
According to another aspect of the present invention is a method for the culturing of functional bone cells, the method comprising applying a suspension of bone cells in physiological media to a synthetic sintered film comprising a stabilized calcium phosphate compound on a substrate; and incubating the bone cells for a period of time to allow expression of bone cell biological activity.
According to a further aspect of the present invention is a kit for monitoring and quantifying the activity of bone cells, the kit comprising a substrate having a sintered film of a stabilized calcium phosphate compound and a multiwell bone cell culture device adhered to the substrate.